International Journal of Molecular Sciences Review Extracellular Vesicles in CNS Developmental Disorders Ana Rita Gomes 1,2,3,4 , Nasim Bahram Sangani 3,4 , Tiago G. Fernandes 1 , M. Margarida Diogo 1 , Leopold M. G. Curfs 4 and Chris P. Reutelingsperger 3,4,* 1 Department of Bioengineering and IBB—Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal; [email protected] (A.R.G.); [email protected] (T.G.F.); [email protected] (M.M.D.) 2 Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina da Universidade de Lisboa, 1649-028 Lisboa, Portugal 3 Department of Biochemistry, Maastricht University, Cardiovascular Research Institute Maastricht, 6200 MD Maastricht, The Netherlands; [email protected] 4 GKC-Rett Expertise Centre, Maastricht University Medical Centre, 6229 ER Maastricht, The Netherlands; [email protected] * Correspondence: [email protected] Received: 18 November 2020; Accepted: 9 December 2020; Published: 11 December 2020 Abstract: The central nervous system (CNS) is the most complex structure in the body, consisting of multiple cell types with distinct morphology and function. Development of the neuronal circuit and its function rely on a continuous crosstalk between neurons and non-neural cells. It has been widely accepted that extracellular vesicles (EVs), mainly exosomes, are effective entities responsible for intercellular CNS communication. They contain membrane and cytoplasmic proteins, lipids, non-coding RNAs, microRNAs and mRNAs. Their cargo modulates gene and protein expression in recipient cells. Several lines of evidence indicate that EVs play a role in modifying signal transduction with subsequent physiological changes in neurogenesis, gliogenesis, synaptogenesis and network circuit formation and activity, as well as synaptic pruning and myelination. Several studies demonstrate that neural and non-neural EVs play an important role in physiological and pathological neurodevelopment. The present review discusses the role of EVs in various neurodevelopmental disorders and the prospects of using EVs as disease biomarkers and therapeutics. Keywords: neurodevelopmental disorders; extracellular vesicles; exosomes; microvesicles; CNS; neurons; astrocytes; glia 1. Introduction 1.1. Extracellular Vesicles Cell-to-cell communication is a fundamental process in coordinating the functions and interactions between the diverse neural cell populations in the central nervous system (CNS) and is mainly organized through secretion of molecules in the intercellular space [1]. Extracellular vesicles (EVs) have been recognized as communication vehicles playing an important role in neural cell proliferation and differentiation, and also in immune modulation and senescence [2]. EVs can be classified and distinguished according to their biogenesis, sub-cellular origin, cargo, size and method of isolation. A subset of EVs, the exosomes, originate from the inward budding of endosomal membranes, giving rise to the formation of multivesicular bodies (MVBs). MVBs typically depict a diameter between 250–1000 nm and contain intraluminal vesicles (ILVs), which are released into the extracellular Int. J. Mol. Sci. 2020, 21, 9428; doi:10.3390/ijms21249428 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 9428 2 of 20 space as exosomes after the fusion of MVBs with the plasma membrane [3]. Exosomes are the smallest EVs and range from 30 to 100 nm in diameter [4]. The microvesicles (MVs) form another subset of EVs. They are larger than exosomes, with a diameter between 0.1 and 1µm. MVs are released from cells by plasma membrane budding [5]. The largest subset of EVs are the apoptotic bodies, which are shed from a dying cell executing apoptosis [6]. The apoptotic bodies can vary in size between 1 and 5 µm in diameter. EVs have been isolated from a great variety of fluids, including supernatants of cultured cells, blood, urine, cerebrospinal fluid (CSF) and serum [7]. Isolation of the different EV subtypes has been accomplished using several methods, such as isolation by size, immunoaffinity capture or precipitation. Isolation by differential ultracentrifugation is widely considered the gold standard method [8–10]. It should be noted, however, that physical and molecular overlap between the EV subsets has precluded the definition of specific EV subtype marker proteins to date [11]. 1.2. Molecular Composition of EVs EVs carry a diverse set of molecules that can be transported over short and long distances to recipient cells. There, they execute defined biological functions, which contribute to health and disease. The composition of EVs is determined by their biogenetic pathway and the microenvironment of the parental cell [12]. The composition may also contribute as a fingerprint for establishing the origin and type of EVs, which is relevant if EVs are to be considered as biomarkers. However, this is not as unambiguous as suggested by many papers on EV research. The endosomal sorting complex required for transport (ESCRT) and accessory proteins are necessary for MVB biogenesis; hence, ESCRT proteins and Alix and TSG101 are considered standard markers of exosomes, regardless of the parental cell type [13]. It has been shown that cells depleted of the ESCRT machinery are still able to produce CD63-positive exosomes by utilizing the sphingomyelinase—ceramide machinery [14]. A recent study with exosomes extracted from neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (hiPSCs) expressed lower levels of Alix, TSG101, Hsp70 and also CD63, in comparison with hiPSC-derived cardiac cells [15]. This might suggest a different protein machinery for neural derived-exosome biogenesis and tracking (i.e., ESCRT-independent pathways) [13]. Other membrane proteins commonly found in exosomes and enriched when compared with cell lysate content are integrins and tetraspanins (such as CD9, CD81, CD82, CD63 and CD37). Exosomes also contain cytosolic proteins, such as heat-shock proteins (Hsp70, Hsp90), in addition to cytoskeletal proteins, like tubulin and actin. Moreover, exosomes contain small GTPases, such as RAB27A, RAB11 and RAB35, which play an important role in intracellular trafficking in secretory pathways during vesicle formation and also in exosome release [16]. Besides the abovementioned typical protein cargo, primary cortical neuron-derived exosomes have been characterized and identified with synaptic proteins, such as L1 cell adhesion molecule (L1CAM), glycosylphosphatidylinositol (GPI)-anchored prion protein and glutamate receptor subunit GluR2/3 [17]. EV types carrying specific neuronal protein cargo will be discussed further in the following sections. Comprehensive analyses of the composition of EV subtypes derived from different cell types revealed a substantial difference in lipidomics and proteomics between exosomes and MVs [17]. MVs encompass more proteasomes, and endoplasmic reticulum and mitochondrial proteins, whereas exosomes express relatively more proteins that function at the interface with the environment [18]. The lipid content also differs between MVs and exosomes. MVs are enriched in ceramides and sphingomyelins, and exosomes carry more glycolipids and free fatty acids [19]. Interestingly, apoptotic bodies have a content that resembles those of cell lysates, yet they also express unique features within their cargo, such as enrichment of thioredoxin peroxidase II, Alix, 14-3-3 and galectin-3 [20]. Apoptotic bodies express more specific surface markers such as phoshatidylserine (annexin A5-binding), thrombospondin and C3b [7,21]. Int. J. Mol. Sci. 2020, 21, 9428 3 of 20 EV types also carry a wide range of genetic material including DNA, mitochondrial DNA (mtDNA), and coding and non-coding RNAs (long non-coding RNAs, micro (mi)RNAs and circular RNAs) [22]. Experimental evidence has demonstrated that the genetic information transferred by EVs can be used by the transcriptional and translational machineries of the recipient cell [22]. Morel et al. identified miR-124a to be abundantly expressed by neuronal exosomes and demonstrated, both in vivo and in vitro, that the neuronal exosomes transfer miR-124a to astrocytes, which consequently upregulated the expression of GLT1 [23]. In a more recent breakthrough, Men et al. have demonstrated that the miRNA profile of secreted exosomes is different from the one observed in live neuronal cells [24]. By generating a cell-type-specific ILVs/exosome reporter (CD63-GFPf/f) in mice, the authors observed that an undescribed neuron-specific miRNA, miR-124-3p, was internalized into astrocytes, also upregulating the glutamate transporter GLT1 [24]. Interestingly, the aforementioned study using vesicles isolated from hiPSC-derived cells from distinct lineage, revealed differential expression of the analyzed miRNA, suggesting the lineage specificity of miRNA cargo. For example, miR-34a-3p was observed to be highly expressed in the hiPSC-derived NPCs, in addition to miR-133a and miR-133b, which may be involved in neurite growth [15]. Nonetheless, miRNAs observed in exosomes from non-neuronal cells, such as mesenchymal stem cells (MSCs), could function as promotors of neurogenesis and neurite remodeling, similar to miR-133b [25]. A significant number of studies have shown the potential of exosomal miRNA as biomarkers, both for diagnostic purposes and for studying several neurodevelopmental and neurodegenerative